Antibodies against p38 (#8690), p-P38 (#4511), ERK1/2 (#4695), p-ERK1/2 (#4376), JNK (#9212), p-JNK (#9255), HNF1α (#89670), HNF4α (#3113) were obtained from Cell Signaling Technology (Cell Signaling Technology, Inc., Danvers, MA, USA). Antibodies against OTUD5 (ab254742), HBc (ab8638), HBx (ab2741), GAPDH (ab181602), Ubiquitin linkage-specific K48 (ab140601), Ubiquitin linkage-specific K63 (ab179434) were obtained from Abcam (Abcam, Cambridge, UK).
Hnf4α
Manufactured by Cell Signaling Technology
Sourced in United States
HNF4α is a transcription factor that regulates the expression of genes involved in glucose, lipid, and drug metabolism. It is a member of the nuclear receptor superfamily and plays a crucial role in the development and function of the liver, kidney, and intestine.
Automatically generated - may contain errors
Lab products found in correlation
Cyclin d1, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Wnt5a, by Abcepta (3 mentions)
Hnf1α, by Cell Signaling Technology (2 mentions)
Metformin, by Merck Group (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Cyclin d2, by Cell Signaling Technology (2 mentions)
P ampk, by Cell Signaling Technology (2 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Ab8638, by Abcam (1 mentions)
Ab2741, by Abcam (1 mentions)
Ab140601, by Abcam (1 mentions)
Ab179434, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Puromycin, by Merck Group (1 mentions)
Cyclin b, by Cell Signaling Technology (1 mentions)
Eif4e, by Cell Signaling Technology (1 mentions)
Nci n87, by American Type Culture Collection (1 mentions)
β catenin, by BD (1 mentions)
P camkii, by Cell Signaling Technology (1 mentions)
12 protocols using hnf4α
1
Detecting Cellular Signaling Pathways
2
Characterization of Gastric Cancer Cell Lines
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We obtained human NCI-N87, AGS and HS 746T tumour cells from the American Type Culture Collection (Manassas, Virginia, USA). The MKN45 GC cell line was made available from Yonsei Cancer Center (Seoul, Korea). SNU-1–5, -16, -620, -216, -484,-601, -638, -668 and SNU-719 GC cell lines were made available from the Korean Cell Line Bank (http://cellbank_snu.ac.kr/ ). Monoclonal antibodies to human hypoxia-inducible factor 1α (HIF-1α) and β-catenin were purchased from BD Transduction Laboratories (San Jose, California, USA); AMPKα, LKB1, HNF4α, antiapoptotic antibody kit (tPARP, cPARP, t-caspase 3, c-caspace 3), t-mTOR, p-mTOR (Ser 2448 and Ser2481), raptor, eIF4E, S6,4E-BP1, WNT1, WNT3, GSK, β-catenin, TCF, LEF, pCAMKII, cyclin A, cyclin B, cyclin D1, cyclin D2 and β-actin were purchased from Cell Signaling Technologies (Boston, Massachusetts, USA) or Santa Cruz Biotechnology (Santa Cruz, California, USA). Antibodies against WNT5A, GSK, β-catenin, pCAMKII, cyclin D2, cyclin D1, cyclin A and cyclin B were purchased from Abgent (San Diego, California, USA), and metformin and puromycin were purchased from Sigma-Aldrich (St Louis, Missouri, USA). SBI-0634689.0001 (BI6015, an HNF4α antagonist) was provided by the Sanford-Burnham Medical Institute (La Jolla, California, USA).
3
Gastric Cancer Cell Lines and Antibodies
4
Immunohistochemical Analysis of Liver Tissue
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Liver tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. The paraffin sections were deparaffinized in xylene and rehydrated by passing through gradually decreasing strengths of ethanol. The sections were then stained with hematoxylin and eosin (H&E) and standard IHC for histopathological analysis. IHC staining was conducted using antibodies against pan CK (1:100; ab234297; Abcam, Cambridge, UK), SOX9 (1:400; #82,630; Cell Signaling Technology, Danvers, MA, USA), Ki-67 (1:400; ab15580; Abcam), YAP (1:100; ab52771; Abcam), TAZ (1:100; #72,804; Cell Signaling Technology), phospho-EGFR (Phospho-Y1068; 1:100; ab40815; Abcam), phospho-MEK1/2 (phospho-Ser217/221; 1:200; #9154; Cell Signaling Technology), phospho-AKT (Phospho-Ser473; 1:400; ab81283; Abcam), phospho-STAT3 (Phospho-Y705; 1:100; #9145; Cell Signaling Technology), HNF4α (1:200; #3113; Cell Signaling Technology), and NOTCH2 (1:100; #5732; Cell Signaling Technology). After incubation with the primary antibodies, the sections were incubated with the appropriate biotinylated secondary antibodies, followed by treatment with freshly prepared DAB substrates (Vector Laboratories, Burlingame, CA, USA).
5
Immunophenotyping of Stem Cells and Hepatocytes
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C3A-iCSCs, D6 and C10 cells were cultured on matrigel for 3 hours before formalin fixation. Permeabilization was performed with 10% Triton® X-100 and blocking with 10% bovine serum albumin (SIGMA-ALDRICH, St. Louis). Primary antibodies against the following molecules were used: NANOG (Abcam, England), OCT4 (Santa Cruz Biotechnology, Dallas), SOX2, SSEA4 (Chemicon, Billerica), TRA-1–81 (Santa Cruz Biotechnology, Dallas), AFP (Abcam, England), ALB (Abcam, England), Hnf4α (Cell Signaling, Danvers), and CD44 (Proteintech, Rocky Hill). Secondary antibodies were Alexa Fluor®488/594goat anti-rabbit/mouse IgGs (Origene, Rockville). Counter staining was performed with Hoechst 33342. Images were captured under a TCSSP5 Confocal Microscope (Leica, Buffalo Grove, USA). Merged images were obtained according to the recommended procedure using the Leica software.
6
Quantitative Analysis of Hepatic TBARS
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TBARS content was measured in liver tissue as previously described.17 (link) Frozen liver tissue (∼50 mg) was homogenized in 500 μL ice-cold cell lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM EGTA, 1 mM EDTA) with phosphatase and protease inhibitors described previously13 (link) using a bead mill homogenizer and ceramic beads. Capillary electrophoresis was performed on Jess Automated Simple Western System (ProteinSimple) according to manufacturer’s instructions with 0.5 mg/mL whole cell lysate and the following antibodies: HNF4α (1:10; Cell Signaling; 31135), BiP (1:20; Cell Signaling; 3183), and vinculin (1:1000; Cell Signaling; 13901). HNF4α and BiP were analyzed using RePlex Simple Western Assay to serially measure protein of interest and loading control (vinculin) within the same assay. Results were analyzed on Compass for Simple Western software.
7
Investigating Berberine Chloride's Anticancer Mechanisms
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Berberine chloride (BBR), dimethyl sulfoxide (DMSO) and Compound C were obtained from Sigma-Aldrich, United States. BI6015 (an HNF4α antagonist) and AICAR (AMPK activator) were provided by the TOCRIS Bio-Techne. Metformin was purchased from Tongji Hospital. Cultrex PathClear Basement Membrane Extract (BME) was obtained from RDsystems. Mouse monoclonal Antibodies against human AMPK, p-AMPK, HNF4α, β-catenin, E-cadherin, cyclin D1, MMP-3, and C-myc were obtained from Cell Signaling Technologies (Boston, MA, United States), WNT5A was purchased from Abgent. Another Antibody against HNF4α (sc-374229) was obtained from Santa Cru Biotechnology (Santa Cruz, CA, United States), which was used for immunohistochemistry of mouse tumor tissue.
8
Western Blotting Protocol for Protein Analysis
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Western blotting was carried out according to a method depicted in the past [45 (link)]. The cells were lysed in the lysis buffer (2% SDS, 5% β-mercaptoethanol, 60 mM Tris-HCl, pH6.8, 0.01% bromphenol blue, and 10% glycerol). The signal was detected using Omni-ECL Femto Light Chemiluminescence Kit (EpiZyme, Shanghai, China). The primary antibodies used in the experiment include β-actin, HNF1α, HNF3α, HNF4α (Cell Signaling Technology, Danvers, MA, USA), and HBc (Synthesized by GenScript, Nanjing, Jiangsu, China).
9
Western Blot Analysis of Hepatic Proteins
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Protein was extracted from frozen organ samples or cultured hepatocytes in cell lysis buffer. Approximately 50 μg of protein was separated on 10% SDS–polyacrylamide gel (SDS-PAGE) gels and transferred to PVDF membranes. Western blot detection was performed using antibodies specific for PIK3R3, GAPDH (Santa Cruz Biotechnology, CA, USA), HNF4α (Cell Signaling Technology, MA, USA), ACADM, CPT1a (Proteintech Group, Chicago, IL, USA), PPARα or PPARγ (Boster, Wuhan, China), which were previously tested and studied using both human and mouse samples.
10
Immunofluorescent Staining of Hepatocytes
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Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature, then remove the PFA and washed with PBS. Next, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. Following washing with PBS, the goat serum was blocked for 30 min and then washed twice with PBS. Cells were then incubated overnight with phalloidin-Alexa 488 (Invitrogen) and primary antibodies against ECAD (1:1000, Cell Signaling), albumin (1:1000, Invitrogen), hepatocyte nuclear factor 4α (HNF4α) (1:50, Cell Signaling) at 4 °C. Finally, the cells were washed with PBS and incubated for 2 h with secondary antibodies Alexa 488 Goat-anti-rabbit (1:500 in blocking solution; Invitrogen). Following washing with PBS, stained cells were imaged in confocal (Zeiss LSM 710) mode. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus for 5 min at room temperature.
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